The regulation of fluid and electrolyte metabolism is a major function of the kidney. Blood enters the kidney through the glomerulus, which filters out cells and proteins and generates, through a process called glomerular filtration, a fluid with an ionic composition identical to that of plasma. The glomerular filtrate then travels through a series of distinct tubular segments, which progressively modify its volume and ionic composition. A large number of factors are known to regulate glomerular filtration including physical forces, local and circulating hormones (Brenner et al. 1976). Similarly, renal tubular reabsorption and secretion are modified by rather complex regulatory processes.
The kidney also serves as an endocrine organ, as it is the main source of erythropoietin, a major determinant of red cell mass that is required for amplification and terminal differentiation of erythroid progenitors and precursors (Line et al., 1985; Jacobs et al., 1985). In addition, the kidney appears to be the most important site for renin release. A fall in blood flow, increased sympathetic stimulation, or a decrease in sodium delivery to the distal tubules can stimulate the release of renin, an enzyme that cleaves angiotensinogen to angiotensin I. The renin-angiotensin system is a key regulator of fluid and electrolyte metabolism, blood pressure, and cardiac function.
Among these many functions carried out by the kidney, the importance of understanding the kidney endocrine function is underscored by the discovery of erythropoietin. There is evidence to suggest that the kidney has complex endocrine functions beyond secretion of renin and erythropoietin. The identification of previously unknown proteins/hormones that are secreted by the kidney will not only provide a more complete understanding of renal physiology but may also significantly improve the way we treat patients with end-stage renal diseases (ESRD).
Patients who develop end-stage renal disease are either treated with renal replacement therapy, such as peritoneal or hemodialysis, or given a renal transplantation. Current renal replacement therapy such as hemodialysis for patients with end-stage renal disease has been the only successful long-term ex vivo organ substitution therapy to date. Despite the success of dialysis at prolonging life, the morbidity and mortality associated with this therapy are undesirably high, and most patients suffer from a poor quality of life (Humes et al., 1995; Wolfe et al. 1999). For example, these patients have increased prevalence of hypertension, cardiovascular diseases such as asymptomatic left ventricular dysfunction, chronic congestive heart failure and atherosclerosis, contributing the most common cause of death among them. While the reasons for this are not entirely clear, it is generally believed that the procedure fails to replicate important functions of the natural organ. The procedure uses an extracorporeal “artificial kidney” to remove excess water and soluble wastes from the blood but does not replicate the important absorptive, metabolic, endocrine, and immunological functions of the natural organ.
It is well documented that patients with ESRD are at significantly higher risk for developing cardiovascular disease, a risk that appears to be correlated with increased oxidative stress (Oberge et al. 2004) and heightened sympathetic tone (Koomans et al., 2004; Joles et al., 2004). Despite the fact that various proteins/hormones have been implicated in ESRD, very few factors involved in ESRD have been identified and characterized. Nevertheless, the identification of such factors is crucial in the development of diagnostics and therapeutics for treatment of ESRD and vascular diseases associated or proteins/hormones secreted by the kidney. Thus, there is long-felt need for the identification and characterization of factors associated with ESRD.
Monoamine oxidase (MAO) is a flavin-adenosine-dinucleotide (FAD)-containing enzyme which converts biogenic amines to their corresponding aldehydes. MAO is present as two isoforms (MAO-A (SEQ ID NO: 11) and MAO-B (SEQ ID NO: 13)), which are separate gene products, that exhibit more than 70% sequence identity and distinct but overlapping substrate specificities in the catabolism of neurotransmitters, such as dopamine, serotonin and norepinephrine (2,3). Both MAO-A and MAO-B are implicated in a large number of neurological disorders and are targets for drugs against Parkinson's disease and depression (4). Mammalian MAOs are bound to the outer mitochondrial membrane and have a FAD molecule covalently bound to the protein via an 8α-thioether linkage to a cysteinyl residue (5). They are expressed in both a tissue-dependent and an age-dependent manner and have been the subject of extensive clinical and pharmacological studies.
MAO-A and MAO-B are anchored through the carboxyl terminus to the outer mitochondrial membrane (Binda et al., 2002). They have overlapping substrate specificity, catabolize neurotransmitters such as epinephrine, norepinephrine, serotonin and dopamine, and are specifically inhibited by pargyline and clorgyline. Polyamine oxidase (PAO), the other known FAD-containing oxidase, is an intracellular oxidase that metabolizes spermine and spermidine, and regulates cell growth (Jalkanen et al., 2001). The crystal structure of human MAO-B has been solved at a resolution of 3.0 A, and reveals a dimer with the FAD cofactor covalently bound to a cysteine side chain (Cys-397) (Binda et al., 2002). MAO-A and MAO-B are coded by adjoining, but separate, genes on the X chromosome, that exhibit over 70% sequence identity and distinct but overlapping substrate specificities in the catabolism of neurotransmitters.
MAO-A and MAO-B differ in tissue distribution, structure and substrate specificity. MAO-A has higher affinity for serotonin, octopamine, adrenaline, and noradrenaline; whereas the natural substrates for MAO-B are phenylethylamine and tyramine. Dopamine is thought to be oxidized by both isoforms. MAO-A and MAO-B are widely distributed in several organs including brain (A. M. Cesura and A. Pletscher, Prog. Drug Research 1992, 38, 171-297). Brain MAO-B activity appears to increase with age. This increase has been attributed to the gliosis associated with aging (C. J. Fowler et al., J. Neural. Transm. 1980, 49, 1-20). Additionally, MAO-B activity is significantly higher in the brains of patients with Alzheimer's disease (P. Dostert et al., Biochem. Pharmacol. 1989, 38, 555-561) and it has been found to be highly expressed in astrocytes around senile plaques (Saura et al., Neuroscience 1994, 70, 755-774). In this context, since oxidative deamination of primary monoamines by MAO produces NH3, aldehydes and H2O2, agents with established or potential toxicity, it is suggested that there is a rationale for the use of selective MAO-B inhibitors for the treatment of dementia and Parkinson's disease. Inhibition of MAO-B causes a reduction in the enzymatic inactivation of dopamine and thus prolongation of the availability of the neurotransmitter in dopaminergic neurons. The degeneration processes associated with age and Alzheimer's and Parkinson's diseases may also be attributed to oxidative stress due to increased MAO activity and consequent increased formation of H2O2 by MAO-B. Therefore, MAO-B inhibitors may act by both reducing the formation of oxygen radicals and elevating the levels of monoamines in the brain.
Given the implication of MAO-B in the neurological disorders mentioned above, there is considerable interest to obtain potent and selective inhibitors that would permit control over this enzymatic activity. The pharmacology of some known MAO-B inhibitors is for example discussed by D. Bentue-Ferrer et al. in CNS Drugs 1996, 6, 217-236. Whereas a major limitation of irreversible and non-selective MAO inhibitor activity is the need to observe dietary precautions due to the risk of inducing a hypertensive crisis when dietary tyramine is ingested, as well as the potential for interactions with other medications (D. M. Gardner et al., J. Clin. Psychiatry 1996, 57, 99-104), these adverse events are of less concern with reversible and selective MAO inhibitors, in particular of MAO-B.
By inhibiting MAO activity, MAO inhibitors can regulate the level of monoamines and their neurotransmitter release in different brain regions and in the body (including dopamine, norepinephrine, and serotonin). Thus, MAO inhibitors can affect the modulation of neuroendocrine function, respiration, mood, motor control and function, focus and attention, concentration, memory and cognition, and the mechanisms of substance abuse. Inhibitors of MAO have been demonstrated to have effects on attention, cognition, appetite, substance abuse, memory, cardiovascular function, extrapyramidal function, pain and gastrointestinal motility and function. The distribution of MAO in the brain is widespread and includes the basal ganglia, cerebral cortex, limbic system, and mid and hind-brain nuclei. In the peripheral tissue, the distribution includes muscle, the gastrointestinal tract, the cardiovascular system, autonomic ganglia, the liver, and the endocrinic system.
MAO inhibition by other inhibitors have been shown to increase monoamine content in the brain and body. Regulation of monoamine levels in the body have been shown to be effective in numerous disease states including depression, anxiety, stress disorders, diseases associated with memory function, neuroendocrine problems, cardiac dysfunction, gastrointestinal disturbances, eating disorders, hypertension, Parkinson's disease, memory disturbances, and withdrawal symptoms.
It has been suggested that cigarette smoke may have irreversible inhibitory effect towards monoamine oxidase (MAO). Boulton et al., “Biogenic Amine Adducts, Monoamine Oxidase Inhibitors, and Smoking,” Lancet, 1(8577): 114-155 (Jan. 16, 1988), reported that the MAO-inhibiting properties of cigarette smoke may help to explain the protective action of smoking against Parkinson's disease and also observed that patients with mental disorders who smoke heavily do not experience unusual rates of smoking-induced disorders. It was suggested that smoking, as an MAO inhibitor, may protect against dopaminergic neurotoxicity that leads to Parkinson's disease and that the MAO-inhibiting properties of smoking may result in an anti-depressive effect in mental patients.
We have previously reported on our discovery of a new monoamine oxidase which we designated as MAO-C. Alternatively, because it is a protein secreted by the kidney, we also termed it ‘renalase.’ Renalase is a secretory form of monoamine oxidase that metabolizes biogenic monoamines such as norepinephrine, dopamine and epinephrine. For further information on the identification and characterization of renalase see, for example, WO 2005/089505, published on Sep. 29, 2005, and Xu et al. (J. Clin. Invest. 115(5):1275-1280 (May 2005), each of which are specifically incorporated by reference herein in their entireties.
The nucleic acid sequence of human renalase (SEQ ID NO: 1) is 27.7% homologous to that of human MAO-A (SEQ ID NO: 10) and 38.2% homologous to that of MAO-B (SEQ ID NO: 12). Renalase has 13% and 12% identity at the amino acid level to MAO-A and MAO-B, respectively. It also has a distinct substrate specificity and inhibitor profile to that of MAO-A and MAO-B, indicating that it represents a brand new class of unique FAD-containing monoamine oxidases.
The human renalase gene resides on chromosome 10, contains 9 exons and spans about 300 Kb. The human renalase gene encodes a 315- or 342-amino acid protein that contains an amino-terminal signal sequence, followed by a flavin-adenosine-dinucleotide (FAD)-containing domain and an amino oxidase domain. Tissue Northern blotting studies demonstrated robust expression of renalase in kidney with much lower levels in all other tissues analyzed. In situ hybridization demonstrated the high level of expression of renalase in proximal and distal tubules.
Renalase was highly expressed in in vitro transcription and translation experiments. Human renalase cDNA is translated to produce a protein with a molecular mass of approximately 38-kDa, which is in agreement with the predicted protein size. Western blotting studies using conditioned medium from transfected HEK293 cells indicates that renalase is a secreted protein. Renalase is present in the plasma at a concentration of 5-10 mg/l in healthy individual.
End-Stage Renal Disease (ESRD) is associated with elevated catecholamine levels, which in turn leads to a myriad of conditions, diseases and disorders, including, for example, asymptomatic left ventricular dysfunction, chronic congestive heart failure and atherosclerosis. These conditions, diseases and disorders are a common cause of death among ESRD patients.
Renalase metabolizes catecholamines in the following rank orders: dopamine>epinephrine>norepinephrine. Renalase is virtually undetectable in patients with ESRD. Thus, the loss or reduced levels of renalase in ESRD patients is at least in part responsible for elevated plasma catecholamine levels, which leads to increased cardiovascular disease, which is a common cause of death among ESRD patients.
In addition, the correlation between renalase levels and renal function make renalase an ideal candidate for a diagnostic marker for renal disease, especially for acute tubular necrosis, a common occurrence in the Intensive Care Unit setting.
The present invention demonstrates that renalase may serve as a therapeutic protein in various forms and in various methods of administration. This invention provides the first demonstration that renalase exists within a mammal in both an active and an inactive form. The inventors have discovered that inactive renalase circulates in the blood waiting to be converted to active renalase that can metabolize biogenic monoamines as necessary.